The goal of this proposal is to understand the physiological mechanisms responsible for adaptation to background light and to bleaches in a mammalian photoreceptor, and the processes that produce photoreceptor degeneration in ocular diseases such as vitamin A deprivation, certain forms of Lebers amaurosis, and retinitis pigmentosa. We will use the techniques of suction-electrode recording and laser spot microscopy with fluorescent indicator dyes on single, isolated rods from mice which, in collaboration with molecular biologists, have been genetically engineered either to lack certain transduction proteins such as the guanylyl cyclase activating proteins (GCAPs) or the RPE65 retinal isomerase, or to contain transduction proteins with site-directed mutations, such as phosphodiesterase gamma T35A. In some experiments recordings will be made from rods in which critical proteins of the transduction cascade are either over or under expressed. Our first specific aim addresses directly one of the program goals of the National Eye Institute to "use both molecular and physiological approaches to study light adaptation in photoreceptors". We shall attempt to discover and delimit mechanisms responsible for changes in rod response waveform and sensitivity in background light, including GCAP-dependent Ca2+/cyclase feedback, modulation of TaGTP hydrolysis rate, and regulation of rhodopsin lifetime via recoverin. Our second specific aim addresses another of the program goals of the NEI to study adaptation in photoreceptors "with particular emphasis on the visual cycle". We will make the first quantitative measurements of adaptation of mammalian rods to pigment bleaching and test the possibility that all-trans retinal released by bleaching light blocks channels of the rod outer segment under physiological conditions. Our third specific aim addresses another of the program goals of the NEI to "determine the pathophysiological mechanisms underlying inherited and retinal degenerative diseases". We will explore possible mechanisms of retinal degeneration during continuous activation, as occurs in vitamin A deprivation and some forms of Lebers amaurosis and retinitis pigmentosa, including oxygen toxicity and prolonged exposure to low free-Ca2+. We shall attempt to define the role of Ca2+ in triggering apoptosis in photoreceptors in rods in which either the cyclic nucleotide-gated channels or the Na+/Ca2+-K+ transporters have been over-expressed or under-expressed, in order to define the relationship between outer segment Ca2+ concentration and the rate of photoreceptor degeneration. PUBLIC HEALTH RELEVANCE: The great majority of diseases of the retina are caused by disorder or degeneration of the photoreceptors, the cells in the eye which convert light into an electrical signal. This proposal seeks to understand basic mechanisms of photoreceptor function, with a view to explaining why vision is abnormal in hereditary night blindness and certain forms of inherited retinal degeneration. Only by understanding why the photoreceptors behave abnormally in these diseases can we hope to learn how to cure them and restore normal vision.